Crystal Structure of Escherichia coli Glucose-1-Phosphate Thymidylyltransferase (RffH) Complexed with dTTP and Mg2+

The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of therffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. InEscherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg2+, refined at 2.6 Å to an R-factor of 22.3% (R free = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall α/β fold and consists of two domains, a larger nucleotide binding domain (residues 1–115, 222–291) and a smaller sugar-binding domain (116–221), with the active site located at the domain interface. The Mg2+ ion is coordinated by two conserved aspartates and the α-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg2+ and leads to a proposal for the dynamics along the reaction pathway. The enzyme glucose-1-phosphate thymidylyltransferase (RffH), the product of therffh gene, catalyzes one of the steps in the synthesis of enterobacterial common antigen (ECA), a cell surface glycolipid found in Gram-negative enteric bacteria. InEscherichia coli two gene products, RffH and RmlA, catalyze the same enzymatic reaction and are homologous in sequence; however, they are part of different operons and function in different pathways. We report the crystal structure of RffH bound to deoxythymidine triphosphate (dTTP), the phosphate donor, and Mg2+, refined at 2.6 Å to an R-factor of 22.3% (R free = 28.4%). The crystal structure of RffH shows a tetrameric enzyme best described as a dimer of dimers. Each monomer has an overall α/β fold and consists of two domains, a larger nucleotide binding domain (residues 1–115, 222–291) and a smaller sugar-binding domain (116–221), with the active site located at the domain interface. The Mg2+ ion is coordinated by two conserved aspartates and the α-phosphate of deoxythymidine triphosphate. Its location corresponds well to that in a structurally similar domain of N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Analysis of the RffH, RmlA, and GlmU complexes with substrates and products provides an explanation for their different affinities for Mg2+ and leads to a proposal for the dynamics along the reaction pathway. Many of the currently available antibiotics target enzymes involved in the synthesis of bacterial cell wall components. Lipopolysaccharides (LPS) are unique and complex glycolipids embedded in the outer membrane of Gram-negative bacteria. They are made of galactose, mannose, rhamnose, 4-acetamido-4, 6-dideoxyglucose, andN-acetylglucosamine. LPS consists of three structural domains, namely the hydrophobic lipid A, the core oligosaccharide, and the O-antigenic polysaccharide (O-PS) (1Godfroid F. Cloeckaert A. Taminiau B. Danese I. Tibor A. de Bolle X. Mertens P. Letesson J.J. Res. Microbiol. 2000; 151: 655-668Crossref PubMed Scopus (71) Google Scholar). Surface polymers have essential roles in the survival of bacteria with the enzymes involved in their formation often found critical to virulence (2Finlay B.B. Falkow S. Microbiol. Mol. Biol. Rev. 1997; 61: 136-169Crossref PubMed Scopus (1182) Google Scholar) and potentially a source of novel targets for therapeutic intervention. The enterobacterial common antigen (ECA) is a unique cell surface glycolipid that is present in all gram–negative enteric bacteria (3Kuhn H.M. Meier-Dieter U. Mayer H. FEMS Microbiol. Rev. 1988; 4: 195-222Crossref PubMed Google Scholar). The genes involved in ECA synthesis cluster near the rfflocus. The product of the rffh gene is a glucose-1-phosphate thymidylyltransferase (EC 2.7.7.24) that catalyzes the reaction that combines dTTP with α-d-glucose 1-phosphate (G-1-P) 1The abbreviations used are: G-1-P, glucose 1-phosphate; GlmU, N-acetylglucosamine-1-phosphate uridylyltransferase; NDP, nucleoside diphosphate; dTTP, deoxythymidine triphosphate; RffH, glucose-1-phosphate thymidylyltransferase; rms, root-mean-squares; dTDP, dioxythymidine diphosphate to yield pyrophosphate and dTDP-glucose. This reaction constitutes the first step in the synthesis of l-rhamnose, a component of the cell walls of both Gram-negative and Gram-positive bacteria (4Shibaev V.N. Adv. Carbohydr. Chem. Biochem. 1986; 44: 277-339Crossref PubMed Scopus (100) Google Scholar). In the Escherichia coli K12 genome, the rffh gene is paralogous to the rfba gene that encodes protein RmlA. The rfba gene is contained within the rfb gene cluster responsible for the synthesis of O-antigen (5Reeves P.R. Hobbs M. Valvano M.A. Skurnik M. Whitfield C. Coplin D. Kido N. Klena J. Maskell D. Raetz C.R. Rick P.D. Trends Microbiol. 1996; 4: 495-503Abstract Full Text PDF PubMed Scopus (418) Google Scholar), and its product RmlA catalyzes the same reaction as RffH. This duplication of function is reflected in a ∼68% amino acid sequence identity between these two enzymes. In addition, a second pair of closely related genes that encode two dTDP-d-glucose 4,6-dehydratases are also present in both clusters: rffg(rff cluster) and rfbb (rfb cluster). The presence of closely related genes in the rff andrfb clusters (both involved in the biosynthesis ofO-polysaccharides) is not unique for these two families (5Reeves P.R. Hobbs M. Valvano M.A. Skurnik M. Whitfield C. Coplin D. Kido N. Klena J. Maskell D. Raetz C.R. Rick P.D. Trends Microbiol. 1996; 4: 495-503Abstract Full Text PDF PubMed Scopus (418) Google Scholar,6Marolda C.L. Valvano M.A. J. Bacteriol. 1995; 177: 5539-5546Crossref PubMed Google Scholar). A similar duplication of functions has been reported for the GDP-mannose biosynthesis genes rfbM and rfbK with genes cpsB and cpsG from the cps cluster involved in the biosynthesis of colonic acid (7Stevenson G. Lee S.J. Romana L.K. Reeves P.R. Mol. Gen. Genet. 1991; 227: 173-180Crossref PubMed Scopus (37) Google Scholar). The rfb cluster also encodes three other enzymes involved in the l-rhamnose synthesis pathway, rmlB, rmlC, andrmlD (8Tsukioka Y. Yamashita Y. Oho T. Nakano Y. Koga T. J. Bacteriol. 1997; 179: 1126-1134Crossref PubMed Google Scholar). The l-rhamnose biosynthetic pathway is not found in mammals, which makes these enzymes potential targets for development of antibacterial drugs. The structure of RmlB has also been described recently (9Allard S.T. Giraud M.F. Whitfield C. Graninger M. Messner P. Naismith J.H. J. Mol. Biol. 2001; 307: 283-295Crossref PubMed Scopus (108) Google Scholar, 10Allard S.T. Beis K. Giraud M.F. Hegeman A.D. Gross J.W. Wilmouth R.C. Whitfield C. Graninger M. Messner P. Allen A.G. Maskell D.J. Naismith J.H. Structure. 2002; 10: 81-92Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Recently, three-dimensional structures of three enzymes belonging to the glucose-1-phosphate thymidylyltransferase family and their complexes with substrate(s), products, and inhibitors have been reported. These include RmlA from Pseudomonas aeruginosa(11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar), RmlA from E. coli (12Zuccotti S. Zanardi D. Rosano C. Sturla L. Tonetti M. Bolognesi M. J. Mol. Biol. 2001; 313: 831-843Crossref PubMed Scopus (93) Google Scholar), and RmlA from Salmonella enterica LT2 (13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google Scholar). These studies revealed high structural similarity among these enzymes, localized their substrate binding sites, defined the arrangement of the substrates and the product in the active site, and showed that dTTP binds to the enzyme in a strained conformation. While the kinetic mechanism of these enzymes is now well established to follow a sequential ordered bi-bi mechanism (12Zuccotti S. Zanardi D. Rosano C. Sturla L. Tonetti M. Bolognesi M. J. Mol. Biol. 2001; 313: 831-843Crossref PubMed Scopus (93) Google Scholar, 13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google Scholar, 14Sheu K.F. Richard J.P. Frey P.A. Biochemistry. 1979; 18: 5548-5556Crossref PubMed Scopus (76) Google Scholar) and proceeds by a S n2 nucleophilic attack of the phosphoryl group of glucose 1-phosphate at the α-phosphate of dTTP (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar), these studies have not clarified the role in catalysis of a strictly required Mg2+ ion (12Zuccotti S. Zanardi D. Rosano C. Sturla L. Tonetti M. Bolognesi M. J. Mol. Biol. 2001; 313: 831-843Crossref PubMed Scopus (93) Google Scholar, 15Bernstein R.L. Robbins P.W. J. Biol. Chem. 1965; 240: 391-397Abstract Full Text PDF PubMed Google Scholar). Neither Mg2+ nor Mn2+ ions were observed in the crystal structures of P. aeruginosa or E. colienzymes despite the co-crystallization efforts, which at the same time showed that this ion is not necessary for binding of the first substrate, dTTP. In the structure of the S. enterica enzyme (13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google Scholar) a feature in the electron density map was interpreted as a Mg2+ ion. However, this ion contacts only the β-phosphate of dTTP and would make no contacts with the phosphate of the second substrate, glucose 1-P (13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google Scholar). Since the other two structures showed that dTTP binding does not require Mg2+, the strict requirements for this ion in catalysis can not be explained by its observed position in the S. enterica structure. Therefore, the binding site of Mg2+ and its precise role in catalysis is still uncertain in this family of enzymes. RffH and RmlA belong to the superfamily of glucose-1-phosphate NTP-transferases, which also includes bacterial uridylyltransferases (e.g. GalF, GalU) and adenylyltransferases (e.g. GlgC). The enzymes from this superfamily in E. coli show 22–26% sequence identity within the ∼240-residue-long catalytic domain compared with RffH. RffH also shows low sequence identity (19% for 230 amino acids) withE. coli N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). Indeed, the catalytic domains of all these enzymes share the same fold and have conserved key catalytic residues. All of them require Mg2+ for catalysis, therefore indicating a common catalytic mechanism. Yet, whereas no metal ion has been identified in the structures of RmlA enzymes, the three-dimensional structures of GlmU from various sources complexed with the NDP-sugar product in the presence of either 10 mm Mg2+ (16Kostrewa D. D'Arcy A. Takacs B. Kamber M. J. Mol. Biol. 2001; 305: 279-289Crossref PubMed Scopus (85) Google Scholar) or 2–10 mm Co (17Olsen L.R. Roderick S.L. Biochemistry. 2001; 40: 1913-1921Crossref PubMed Scopus (126) Google Scholar) contain a well ordered metal ion. We have succeeded in obtaining crystals of RffH from E. coliin the presence of deoxythymidine triphosphate (dTTP) and Mg2+, with both ligands clearly visible in the electron density maps. The location of the Mg2+ ion in our structure differs from that described in S. enterica RmlA, and in combination with the previously determined structures of RmlA complexes from the three species, our data provide a clear explanation for the essential role of Mg2+ in the catalytic mechanism of these enzymes. This Mg2+-binding site coincides with that observed in GlmU enzymes, providing additional evidence for a common catalytic mechanism. Comparison of all available structures allows us to make hypotheses regarding conformational flexibility at the active site during catalysis. The rffH gene was cloned into a derivative of the pET-15b vector (Amersham Biosciences) containing a thrombin cleavage site to obtain an in-frame N-terminal fusion with His6. Plasmid DNA was transformed into E. coli DL41 (18Hendrickson W.A. Horton J.R. LeMaster D.M. EMBO J. 1990; 9: 1665-1672Crossref PubMed Scopus (1008) Google Scholar) for selenomethionine protein production. The transformed bacteria were grown at 37° to anA 600 of ∼0.8 in defined LeMaster medium supplemented with 25 mg/liter of l-selenomethionine. A 1 liter culture was induced with 100 μmisopropyl-1-thio-β-d-galactopyranoside (IPTG) and the culture continued at room temperature for an additional 15 h. Cells were harvested by 25 and in of mm mm 10 mm in which one of inhibitors was Cells were by for a of with between The was by The protein was first an (Amersham Biosciences) and the were a acid The was with mm and the bound with containing mm RffH as a both and and its in was by a of RffH were with the in the presence of protein was to 10 by and by A of of protein in mm mm mm was with of containing acid and and the to the of in data to 2.6 Å were at an a The These crystals belong to group with a = = = and = and was the and W. 1997; PubMed Scopus Google Scholar). The crystal structure was determined by the with the J. Scopus Google Scholar) the of RmlA from P. aeruginosa (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar) as the showed as in the The the a of and = was with the Biol. PubMed Scopus Google Scholar) to the of the electron density of map with the M. 1991; Scopus Google Scholar) and the P.D. P. Jiang J.S. J. M. T. D. PubMed Scopus Google Scholar) to The R-factor is (R free = for all data (R = and free = for data with to 2.6 Å The is of as by the low for and from target and the one is found in the This is located in a and is well defined in the electron density of RffH have been to the with and of the protein = = = for a of of not in the in a = = = for a of of not in the The structure of RffH was determined by the RmlA structure aeruginosa (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar) as a are two of RffH in the The is present in the protein used for one from the is visible in the density and is in the The two are The in the two RffH of from to for one Mg2+ one dTTP and a of are in I. The RffH to the α/β fold The overall of the monomer is the RffH structure is similar to that of RmlA (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar, S. Zanardi D. Rosano C. Sturla L. Tonetti M. Bolognesi M. J. Mol. Biol. 2001; 313: 831-843Crossref PubMed Scopus (93) Google Scholar, 13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google Scholar) not the structure in provide only a The is from that into a with and one and both of the The is with an of three two additional are one along the of the and the other near the of the data of RffH at a protein of the presence in of a with a of This corresponds to a tetrameric of the similar to that of RmlA from E. P. and S. The arrangement of in the crystal clearly shows the presence of in the a dimer with the to the and closely with a dimer along into a Therefore, the has an The of the is The surface of monomer A is of which is in with monomer and contacts the monomer In of the monomer surface is for of a monomer of RffH and RmlA an of Å Å and Å for E. P. and S. Comparison of the RffH with of RmlA shows that a of these is that of of A and have a larger A and related to and this dimer well with the of of the three RmlA structures of however, a of in the and Å of the within a RffH with RmlA. This of the arrangement has the dTTP binding site found in RmlA that is located at the between the two is no available for binding dTTP in the RffH The of an which in RmlA a with a and provides a surface for the is in RffH into the position by dTTP in the with the In the complex the dTTP binds at the of the and of the The triphosphate a to the of the with the α-phosphate and β-phosphate in an indicating conformational The phosphate makes with the for its conformational The is well and binds in a by the which the conserved This sequence is also conserved in the related GlmU and and is similar to that found in the two to the group of one to and a to an of and to A is between the and of the a to a potentially involved in catalysis (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar). This of dTTP is similar to that observed in the RmlA In the E. coli RmlA complex with the reaction a ion is located in the same as of dTTP, indicating high of this binding for The of a is by with the The makes one with of the The of the is also to the of and This the of with triphosphate the amino group the in a of a to the The a to the of from the same that binds the triphosphate. is also a between the of and the of of the various RmlA complexes with bound dTTP clearly shows that the binding of this substrate is the same in all of these enzymes. The electron density map for RffH shows density near the of dTTP in monomer located Å from and the that this is a bound Mg2+ ion. of bound unique for structures of RmlA enzymes and is by the high of in the protein ligands in the are: an from of dTTP, two from the one from the and one The the of the is However, was not clearly visible in the electron density at this The two aspartates the Mg2+ in the are strictly conserved in the from the family The position of the Mg2+ ion corresponds to the position of a metal ion in the structures of GlmU complexed with the the that this is the ion. the same the of a of Mg2+ for binding as compared with GlmU a for the The position of the Mg2+ ion observed is different from that in the structure of S. enterica RmlA this ion is bound near of dTTP, the to the binding site of This ion is coordinated by the and by the of a not conserved in other The location of this Mg2+ ion from the binding site, the of of the providing and a makes this binding site to to that for the essential Mg2+ ion. is well established that catalysis in the RmlA family S n2 nucleophilic attack of a phosphoryl of at of dTTP and the presence of Mg2+ ions (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar, S. Zanardi D. Rosano C. Sturla L. Tonetti M. Bolognesi M. J. Mol. Biol. 2001; 313: 831-843Crossref PubMed Scopus (93) Google Scholar, 13Barton W.A. Lesniak J. Biggins J.B. Jeffrey P.D. Jiang J. Rajashankar K.R. Thorson J.S. Nikolov D.B. Nat. Struct. Biol. 2001; 8: 545-551Crossref PubMed Scopus (119) Google R.L. Robbins P.W. J. Biol. Chem. 1965; 240: 391-397Abstract Full Text PDF PubMed Google Scholar). The presence of two conserved that as ligands to the Mg2+ ion in our the essential Mg2+ binding site for this family of enzymes. of RmlA with bound 1-phosphate the structure of RffH with bound dTTP and Mg2+ shows that the phosphate group 1-phosphate is in to the Mg2+ ion and to the α-phosphate The presence of the Mg2+ ion at the position observed in our structure would provide between the two phosphate binding of G-1-P, with of both in the of this ion as of a to the Mg2+ ion by one of the of the would the of this of the of would the α-phosphate of dTTP between the three well for nucleophilic with this a metal either (17Olsen L.R. Roderick S.L. Biochemistry. 2001; 40: 1913-1921Crossref PubMed Scopus (126) Google Scholar) or Mg2+ (16Kostrewa D. D'Arcy A. Takacs B. Kamber M. J. Mol. Biol. 2001; 305: 279-289Crossref PubMed Scopus (85) Google Scholar), has been observed to the and phosphoryl of the product complexed with In the structures of complexes of RmlA with either or dTTP in the of bound Mg2+ the position of its is whereas that of is found in two different either a with an to of or a to to of of GlmU and RffH coli that the position of the first is whereas the second is an in a different location the second in RffH. This the metal at a different position within the in GlmU these two are found in the same position in all of the presence or of substrate or The of the Mg2+ binding site in GlmU with the site in and is reflected by in for Mg2+ in these two enzymes. Comparison of the structures of RmlA and GlmU complexed with the product or shows a in the of the and the with two of in the position of the phosphoryl group to the Its position is to either the metal ion one or a the other In of these two is one between the and they the of the phosphate In the RmlA enzymes these two are by a that is also to an In the GlmU enzymes, the two the are ligands of the metal ion Comparison of the complexes containing shows a similar same of the 1-phosphate between two This of the position of the phosphate group to the that this group during of the of reaction from a complex with RmlA and from the complex with GlmU and residues. The is the nucleotide of the The are to RmlA. reaction substrate glucose 1-phosphate complexed with E. coli RmlA and P. to The kinetic data for an ordered bi-bi kinetic mechanism. The binds first in the active This is with nor the presence of Mg2+, is by of the with the conserved sequence in and by contacts made by the nucleotide In this at three along the phosphate are into the of the its this can with two of the both by a of We that in the presence of bound the binds with its phosphate from the α-phosphate of of the second substrate the Mg2+ binding The of the Mg2+ ion a of its in RmlA and a of the phosphoryl group of the to the ion. The of the phosphate necessary to the the second phosphate for an attack the α-phosphate the to In to these the Mg2+ would to the required for formation of the by the as for GlmU (16Kostrewa D. D'Arcy A. Takacs B. Kamber M. J. Mol. Biol. 2001; 305: 279-289Crossref PubMed Scopus (85) Google Scholar). of pyrophosphate with the formation of the to the with an of the The of the diphosphate of the product Mg2+ and leads to its with a of the phosphate bound to the into an with a of the product be by the of the to the now site by the The proposal that of P. aeruginosa RmlA a key in catalysis by to the group of dTTP (11Blankenfeldt W. Asuncion M. Lam J.S. Naismith J.H. EMBO J. 2000; 19: 6652-6663Crossref PubMed Scopus (162) Google Scholar) in of our structural for RffH. our the function of and in related enzymes is to Mg2+, which in would catalysis by a combination of of the the of the group and the 1-phosphate of the second substrate for nucleophilic We for of the E. coli rffh gene, for with data for the and D. for

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